G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that are involved in many aspects of pathophysiological processes. There are approximately 800 GPCRs in the human genome, and about 30 GPCRs have been validated as therapeutic targets that account for more than 30 percent of marketed drugs for a variety of diseases (Jacoby E. et al. 2006, ChemMedChem Vol. 1, pp. 761-782). However, hundreds of GPCRs remain to be characterized and explored as potential pharmaceutical targets. There is a significant interest in defining the function of the GPCRs in physiology and pathological conditions and then modulating the GPCR signaling pathways for the treatment of diseases. Drug discovery efforts have been concentrated on identifying compounds that either stimulate or inhibit GPCR activities.
GPR4 (Gene ID: 2828), a member of the proton-sensing GPCR family, has recently been identified as a novel pH sensor (Ludwig M. G. et al. 2003, Nature Vol. 425, pp. 93-98; Yang L. V. et al. 2007, Mol. Cell. Biol. Vol. 27, pp. 1334-1347; Liu J. P. et al. 2010, Pharmacol Res. Vol. 61, pp. 499-505). GPR4 encodes a cell surface receptor consisting of 362 amino acids and is expressed in a wide range of tissues such as the lung, kidney, heart, intestine, and liver. GPR4 is highly conserved during evolution, with more than 90% protein sequence homology among mammalian orthologs and more than 70% homology between human and zebrafish orthologs. However, the biological function of GPR4 is not clearly defined. GPR4 was previously reported to be a receptor for the lysolipids sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), but this observation has not been confirmed and the original publication has been retracted (Zhu K. et al. 2001, J Biol Chem Vol. 276, 41325-41335. Retraction, 2005, Vol. 280, 43280). Several studies indicated that GPR4 mediates the SPC-induced endothelial tube formation, LPC-induced impairment of endothelial barrier function, and LPC-induced VCAM-1 expression (Kim K. S. et al. 2005, Faseb J. Vol. 19, pp. 819-821; Qiao J. et al. 2006, Am J Physiol Lung Cell Mol Physiol. Vol. 291, pp. L91-101; Zou Y. et al. 2007, FEBS J. Vol. 274, pp. 2573-2584). However, as the ligand-receptor relationship between SPC,
LPC and GPR4 is not validated, it is unclear whether GPR4 directly or indirectly mediates the biological effects of the lysolipids SPC and LPC.
More recent studies demonstrate that GPR4 predominantly functions as a proton sensor activated by extracellular acidic pH (Ludwig M. G. et al. 2003, Nature Vol. 425, pp. 93-98; Yang L. V. et al. 2007, Mol. Cell. Biol. Vol. 27, pp. 1334-1347; Liu J. P. et al. 2010, Pharmacol Res. Vol. 61, pp. 499-505). Several extracellular histidine residues of GPR4 are important for the proton sensing and the receptor activation. Acidosis-induced GPR4 activation stimulates the Gs/cAMP signaling in endothelial cells and regulates the growth of microvessels (angiogenesis) (Yang L. V. et al. 2007, Mol. Cell. Biol. Vol. 27, pp. 1334-1347). Since blood vessels form and/or function in acidic tissue microenvironments in many pathological conditions such as inflammation, ischemia and cancer, it is very important to delineate how acidosis regulates the growth and function of blood vessels, by which new therapeutic opportunities may be provided. It should be pointed out that the growth of new blood vessels (i.e. angiogenesis) and the function of blood vessels (e.g. vascular adhesion and vessel tone) are distinct aspects of vascular biology and involved in different diseases.
Local or systemic acidosis is associated with a variety of pathological conditions such as inflammation, ischemia, tumor, diabetic ketoacidosis, and lung and renal diseases due to defective blood flow, hypoxia, and glycolytic metabolism (Nedergaard M. et al. 1991, Am J Physiol. Vol. 260, R581-588; Hunt J. F. et al. 2000, Am J Respir Crit Care Med. Vol. 161, pp. 694-699; Lardner, A. 2001, J Leukoc Biol. Vol. 69, pp. 522-530). For instance, interstitial pH in ischemic organs often decreases to 7.0-6.0 and sometimes below 6.0. Acidosis has profound effects on blood vessels, immune cells, inflammatory responses and tissue injury, but it is previously unknown whether GPR4 regulates vascular endothelium-leukocyte interaction and inflammation in response to acidosis.
The interaction between vascular endothelial cells and blood cells plays critical roles in inflammatory responses and thrombosis. Adhesion of leukocytes to endothelium is an important early step through which immune cells extravasate into inflammatory tissues to perform effector function. In the microenvironment, endothelial cells and blood cells are activated to trigger an inflammatory phenotype such as the increase of adhesion molecules and secretion of cytokines and chemokines. Inhibition of endothelium-blood cell interaction has been pursued as therapeutic approaches to treat inflammation (Stefanelli T. et al. 2008, Autoimmun Rev. Vol. 7, pp. 364-369). In the present invention, GPR4 is identified as a new therapeutic target for inhibiting acidosis-induced inflammatory responses of endothelial cells and the related methods for the inhibition are provided. It would be an advance in the art to provide method to treat inflammation in acidosis-related diseases.